Comparison of ertapenem non-susceptibility with 2-mercaptopropionic acid phenotypic tests in predicting NDM-1 and IMP-1 production in clinical isolates of Escherichia coli

Background: A routine phenotypic test has not been recommended for the detection of metallo-β-lactamases (MBLs) producing Enterobacteriaceae species such as Escherichia coli. The current study was conducted to compare the 2-mercaptopropionic acid (2-MPA) phenotypic method and ertapenem non-susceptibility test with polymerase chain reaction in predicting the production of MBLs in clinical isolates of E. coli. Methods: Antimicrobial susceptibility test for beta-lactam antibiotics were performed by disk diffusion method. All isolates which showed inhibition zones of ≤ 22 mm for CAZ and ≤ 27 mm for CTX were considered potential MBLs producing isolates. The production of MBLs was confirmed using 2-MPA compound. Also, susceptibility to ertapenem was evaluated in all isolates. Conventional PCR was performed to detect blaIMP-1 and/or blaNDM-1 genes in all potential MBLs producing E. coli isolates. Results: Of 259, 138 (53.3%) isolates were potential MBLs producing bacteria. One hundred and fifteen out of 138 (83.3%) isolates were susceptible to ertapenem. MBLs production was confirmed in 75/138 (54.4%) isolates by 2-MPA phenotypic method. The blaNDM-1 or/and blaIMP-1 genes were found in 30/75(40%) and 39/115(33.9%) isolates which were confirmed by 2-MPA and were susceptible to ertapenem, respectively. The sensitivity of 2-MPA method and ertapenem non-susceptibility test compared with PCR were 65.2% and 15.2%, and the specificity was 52.1% versus 82.6%, respectively. Conclusion: This study demonstrated that the 2-MPA phenotypic method does not have acceptable sensitivity and specificity in comparison with PCR, but its results are more reliable for the detection of MBL producing E. coli isolates compared with non-susceptibility to ertapenem.

Carbapenemase-producing Enterobacteriaceae (CPE) hydrolyze carbapenems by obtaining beta-lactamase genes through plasmids. These enzymes are categorized in three Ambler classes (4). The most prevalent identified carbapenemase in Enterobacteriaceae are KPCs (klebsiella pneumoniae carbapenemase), Metallo beta-lactamases (MBLs) including NDM, IMP, VIM and OXA-48-like in A, B and D classes, respectively. MBLs producing bacteria are often resistant to all beta-lactam antibiotics and bacteria which carry bla-NDM genes are the most prevalent carbapenemase producers in Asian countries (5). Several reports indicate that CPE, particularly those carrying various MBLs genes, distribute rapidly throughout the world (2,6,7).
Non-susceptibility to ertapenem is the most sensitive indicator for carbapenemase producing bacteria, as suggested by Clinical and Laboratory Standards Institute (CLSI). Based on our best knowledge, a routine standard phenotypic test for MBLs detection is not used in clinical laboratories. The modified Hodge test (MHT) for the identification of carbapenemase (KPC) in Enterobacteriaceae is recommended by CLSI. As the MHT test has low sensitivity and specificity for the detection of IMP, NDM and the other carbapenenemases, CLSI introduced different tests which are more sensitive and specific, although they are not recommended for routine clinical tests (8).
The efficacy of carbapenems have been documented in the treatment of some infection diseases caused by gram negative bacteria such nosocomial pneumonias. However, it is unclear that this effect is confirmed by all of published studies (9). For example a study which used CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocols on susceptibility to carbapenems showed that about 50% of CPE isolates which are susceptible to carbapenems (MIC assay) did not respond to treatment (10). Moreover, Adler et al. showed that MIC measurements alone may not be adequate in predicting the therapeutic efficacy of carbapenems against CPE. The complex interplay of different factors affect the resistant phenotype which is not identified by routine antimicrobial susceptibility test (AST) testing, therefore it is required to augment with a phenotypic test in order to detect CPE isolates (11).
New phenotypic methods have been developed using different compounds which inhibit metallo-betalactamase activity in bacteria such as 2-mercaptopropionic acid (2-MPA) and ethylenediaminetetraacetic acid (EDTA). Several studies have utilized different compounds and demonstrated that 2-MPA yielded more accurate results than other compounds (12)(13). The current study was conducted to compare the 2-mercaptopropionic acid (2-MPA) phenotypic method and ertapenem non-susceptibility test with polymerase chain reaction (PCR) in predicting the production of MBLs in clinical isolates of E. coli as the most common infectious disease among Enterobacteriaceae throughout the world.

Methods
Bacterial isolates: Two-hundred fifty-nine non-duplicate E. coli isolates were obtained from various clinical specimens of patients admitted to three Babol university of Medical Sciences affiliated hospitals at Babol located in the North of Iran, between June 2015 and November 2016. Morphological features of bacterial colony, gram stain, and biochemical tests were used to identify the isolates as E. coli. Antimicrobial susceptibility test (AST): Disc diffusion assay was carried out on Mueller Hinton agar (MHA) for betalactam antibiotics including cefotaxime (CTX), ceftazidime (CAZ), cefpodoxime (CPD), cefepime (CPM), ertapenem (ETP), aztreonam (ATM) (Mast discs, UK) according to CLSI standard protocol (8). Phenotypic test for MBLs: All isolates which showed inhibition zones of ≤ 22 mm for CAZ and ≤ 27 mm for CTX in the preliminary AST were considered potential MBLs producing E. coli (screening test). These isolates; I, were examined by 2 MPA and CAZ discs to confirm MBLs production (confirmatory test) (14,15) and II, were evaluated for non-susceptibility to ertapenem (resistant or intermediate) and to at least one antibiotic from third-generation cephalosporins according to CLSI, 2016 protocol (8). Molecular analysis: All potential MBLs isolates obtained by screening test were further assessed by PCR as the gold standard test (16). DNA extraction and PCR: Total DNA was extracted from the bacterial suspension in sterilized normal saline using a commercial DNA extraction kit (Cinnagen Co., Iran) according to manufacturer's instruction. Then, PCR analysis of blaIMP-1 and blaNDM-1 genes was performed using a specific paired primer as follows: NDM-1, 5 ʹ ACCGCCTGGACCGATGACCA3ʹ (forward) and 5ʹGCCAAAGTTGGGCGCGGTTG3ʹ (reverse); IMP-1, 5 ʹ TTGACACTCCATTTACAG3ʹ(forward) and 5 ʹ GATTGAGAATTAAGCCACTCT 3ʹ (reverse) (17).

Shahandeh Z, et al.
Acinetobacter baumannii and E. coli K12 were used as positive control strains for blaIMP-1 and blaNDM-1 genes, respectively (18,19). Amplification was carried out as follows: initial denaturation at 94 o C for 10 min; 30 cycles of 94 o C for 40s, 55 o C for 60s and 72 o C for 1min; and a final elongation step at 72 o C for 7min for the blaIMP-1 gene assay. The annealing temperature was optimal at 58 o C for the amplification of the blaNDM-1 gene. The PCR products were electrophoresed on a 2% agarose gel and evaluated by gel documentation system (Vilbert, Lourmat, France). Statistical analysis: Data were analyzed by SPSS (ver.22) using descriptive statistics. The sensitivity and specificity of 2-MPA phenotypic and ertapenem non-susceptibility tests compared with PCR were evaluated.
The sensitivity and specificity of the abovementioned tests for the detection of MBLs compared with PCR are shown in table 2. The sensitivity of 2-MPA method and ertapenem nonsusceptibility test compared with PCR were 65.2% and 15.2%, and the specificity was 52.1% versus 82.6%, respectively.

Discussion
MBLs producing bacteria cause serious problems for the treatment of infectious diseases as they carry the most prevalent transferable carbapenemases which often cause resistance to all beta-lactam antibiotics (1). Generally, the genes responsible for the MBLs production are a part of an integron structure and carried on transferable plasmids or the genome (13). Since, a diverse number of genetic elements responsible for carbapenemase production are present in the bacterial genome, there is no standardized phenotypic test for the detection of MBLs producing bacteria in routine clinical laboratories (16).
Non-susceptibility to ertapenem is the most sensitive indicator for carbapenemase producing bacteria (8). In this study, the sensitivity of non-susceptibility test to ertapenem in comparison to PCR analysis was very low (15.2%) while the specificity of this test was acceptable (82.6%). Also, the 2-MPA phenotypic method had moderate sensitivity and specificity but it was a more sensitive technique in detecting MBLs producing E. coli (DOR=1.96) in comparison with non-susceptibility to ertapenem (DOR=0.85) (table 2). The specificity and sensitivity of the 2-MPA phenotypic method for the detection of IMP enzyme were reported as 100% and 51.1% in K. pneumoniae, E. cloacae and C. freundii by Yan et al. (20). Also, Dobrzaniecka et al. reported that 2-MPA was more effective than EDTA compounds for the detection of MBLs, particularly in Enterobacteriaceae (12). Furthermore, the sensitivity and specificity of automated Microscan Walkaway system compared with non-susceptibility to ertapenem for the detection of IMP and NDM enzymes were 98% and 13%, respectively (21). A possible explanation for these differences obtained in our study and the aforementioned reports may be geographical variation, type of carbapenemase producing genes, bacterial species and sample size. Other findings obtained in the current study showed that 30.4% (7/23) of E.coli isolates diagnosed as potential MBLs based on non-susceptibility to ertapenem (8) carried blaNDM-1 and/or blaIMP-1 genes (table 2). A possible explanation for the lack of amplification of these genes in 69.6% (16/23) of the isolates may be due to existence of other carbapenemase producing genes or the presence of other resistance mechanisms. It is noteworthy that blaNDM-1 and/or blaIMP-1 genes were detected in 33.9% (39/115) of E.coli isolates which were susceptible to ertapenem. This finding is supported by Cetinkol et al., who reported that in spite of a low resistance rate to ETP (2.1%), 22.1% of E.coli isolates carried the blaNDM gene (22). Another study also reported that 18 (2%) isolates which were susceptible to ertapenem or other penems, carried carbapenemase producing genes such as blaNDM and blaIMP (21). Moreover, the existence of these genes in isolates susceptible to ertapenem and imipenem were reported by Karlowsky et al., 2017. Although the presence of carbapenemase producing genes in a low-number of such isolates has been reported, it can cause serious problems in the treatment of infectious diseases. Since penem susceptible isolates carry carbapenemase producing genes may act as reservoirs and can transfer these genes to other pathogens (5).
Aztreonam is a monobactam antibiotic that is not hydrolyzed by MBLs and is active against most strains of gram-negative microorganisms. The co-existence of MBL genes with other beta-lactamase genes including extendedspectrum beta-lactamases (ESBL) on a plasmid breaks down the aztreonam antibiotic (23). The existence of blaCTX-M, blaTEM and blaSHV genes were evaluated in these bacteria and our results showed that 100% of isolated E. coli that were non-susceptible to aztreonam which carried MBL genes, also carried the abovementioned genes. These findings are supported by other studies which demonstrate that 56.8% of isolated E. coli were resistant to aztreonam and also carried ESBL producing genes (24). Furthermore, a study performed in South Korea showed that 73.3% of NDM-producing E. coli was resistant to aztreonam (25). The limitations of our study are listed as follows: (i) antibiotic susceptibility was assessed only by disc diffusion method; (ii) AST was not performed for imipenem and meropenem; (iii) Only blaNDM and blaIMP genes were evaluated for MBLs production.
In conclusion, this study demonstrated that although the 2-MPA phenotypic method does not have acceptable sensitivity and specificity in comparison with PCR, its results are more reliable for the detection MBL producing E. coli isolates compared with non-susceptibility to ertapenem. The current work also recommends that E. coli isolates susceptible to ertapenem should be further evaluated for carbapenemase production by the other phenotypic tests.